The overall goal of this proposal is interrogate gastrointestinal (GI) niche establishment of the diarrheagenic pathogen, Clostridium difficile. We have identified a unique oxygen detoxifying enzyme, superoxide reductase (SOR), which promotes C. difficile survival in the GI tract. The immediate impact of this work will be to validae SOR and other redox proteins as novel targets for interventions designed to prevent C. difficile colonization. Three Specific Aims are proposed. In Aim 1, we will delineate the impact of oxygen and reactive oxygen species (ROS) exposure on C. difficile localization and survival in the GI tract. In Aim 2, we will assess the impact of host ROS deficiency on C. difficile niche establishment. In Aim 3, we will comprehensively map the C. difficile redox-stress response compendium. All the proposed studies will use recent clinical isolates of C. difficile that we have obtained through ongoing disease surveillance studies. For Aims 1 and 2, two animal models will be employed, and bacterial establishment tracked via live-animal and ex vivo imaging using a novel colonization tracer we have specifically developed for these studies. For Aim 3, we will employ next-generation redox proteomics (ROSics) approaches.